Oligosaccharides having anti-Xa activity and pharmaceutical compositions containing them

ABSTRACT

Oligosaccharides obtainable from heparin including heparinic constituents of molecular weights ranging from 2000 to 50,000. Said fractions have a Yin-Wessler titer and a USP titer in a ratio of at least 30. They consist of chains constituted by no more than 8 saccharidic moities. They possess a strong antithrombotic activity and are then useful as antithrombotic drugs.

This application is a continuation-in-part of patent application Ser.No. 091,164, filed Nov. 5, 1979, which application has been abandoned infavor of pending application Ser. No. 204,505 filed Nov. 6, 1980.

BACKGROUND OF THE INVENTION

The invention relates to oligosaccharidic fractions and tooligosaccharides having biological properties, particularly the abilityof more specifically controlling some steps of the blood coagulation.

The invention also relates to processes for obtaining said products andto the use of said products as active principles in drugs.

The invention relates more particularly to oligosaccharides having ahighly selective activity against activated factor X or factor Xa ofblood i.e. a strong antithrombotic activity, while avoiding the risk ofhemorrhage for the patient, as well as to oligosaccharidic fractionscontaining such oligosaccharides (the term "oligosaccharidic fractions"is used in the specification and the claims to designate a relativelyhomogeneous mixture of oligosaccharidic fragments or chains having avariable number of saccharidic moieties).

The inventors have been led to investigate the biologically activeoligosaccharidic fractions and the oligosaccharides themselves, such asobtained from heparin.

It will be noted that the term heparin is used in the specification andthe claims in its broadest sense, in order to designate either acommercial heparin of pharmaceutical grade or a crude heparin such asobtained by extraction from biological material, particularly frommammalian tissue.

It is admitted that heparin is an heterogeneous polysaccharide withrespect to the composition of its oligosaccharidic chains as well as tothe molecular weight thereof.

It is generally considered that heparin mainly contains2-O-sulfate-L-iduronic acid and N-sulfate-D glucosamine (6-O-sulfated ornot) and to a lesser extent D-glucuronic acid, L-iduronic acid andN-acetyl-D-glucosamine (6-O-sulfated or not) moieties.

It is also known that heparin produces its anticoagulant activity bypotentiating the inhibitory effect of antithrombin III (αATIII) which isa plasma protein against the successive enzymatic reactions of thecoagulation cascades. As heparin is able to simultaneously depress alarge number of the coagulation factors participating in the creationand the unkeeping of different forms of hypercoagulability, its activitydoes not appear specific but general.

Although this anticoagulant activity turns out to be valuable, there-equilibration of the coagulation-fibrinolysis system with patientsunder treatment is delicate, due to the global nature of its action. Asa result the administration (in order to prevent hypercoagulation risks,for example, the spectre of post-surgical thrombosis) of too high dosesof anticoagulant drug or the insufficient selectivity of that drug canbe responsible for serious hemorrhages.

SUMMARY OF THE INVENTION

By thoroughly studying various conditions for depolymerizing heparin andthe depolymerization mixtures thus obtained, the inventors have been ledto notice that under certain conditions, it is possible to obtainvaluable antithrombotic oligosaccharides containing mixtures. Theseoligosaccharides are more satisfactory than heparin with regard to thespecificity of their activity. They are more particularly capable ofenhancing the specific activity of ATIII with respect to a smallernumber of coagulation factors, more especially with respect to factorXa. More essentially, it has been found that such fractions andoligosaccharides have a low global anticoagulant activity as measured bythe USP method and that consequently the ratio of their anti-Xa activityas expressed in Yin-Wessler units and of their USP titer is high, atleast 30.

As is well-known, the Yin-Wessler activity is more specificallyrepresentative of the capability of the active fractions to potentiatethe inhibition of the activated factors Xa of blood by AT III in thecorresponding test and the UPS titer is representative of the capabilityof the active fractions to inhibit the global coagulation of blood orplasma.

The Yin-Wessler titer is measured by the test described by these authorsin J. Lab. Clin. Med. 1976,81,298-300, and the U.S.P. titer is measuredby the test which is described in the "Pharmacopea of the United Statesof America", XIX, pp. 229-230 (see also the second supplementU.S.P.--NF, p, 62 and the fourth supplement U.S.P.--NF p. 90respectively entitled "Drug Substances" and "Dosage Forms".

Unexpectedly, it has thus been noticed that the desired anti-Xa specificactivity was found in the short oligosaccharidic chains, i.e. containingno more than 8 saccharidic moieties, which can be isolated from thedepolymerization mixtures by resorting to certain purification steps,carried out under specified conditions.

It will be noted that the terms "saccharidic moiety" is used in thespecification and the claims to designate monosaccharides contained inthe heparinic chains.

Besides, according to an aspect of great interest, the inventors havefound that the anti-Xa activity of said fractions and of theoligosaccharides, such as expressed in Yin-Wessler units, wassignificant of an antithrombotic activity in vivo.

It is then an object of the invention to provide new oligosaccharidesand fractions containing them, having a high anti-Xa activity and aremarkable selectivity in the framework of the successive enzymaticreactions which characterize the coagulation processes. It is anotherobject to provide structural features of these oligosaccharides.

It is a further object of the invention to provide a process ofobtaining said fractions which is easy to carry out.

It is still another object of the invention to provide active principlesof drugs and the drugs per se, particularly capable of inhibiting thefactor Xa with a high degree of selectivity while their activity onglobal coagulation can be maintained at a very low level. Such drugs areadvantageously useful for antithrombotic treatment without haemorrhagerisks.

Said oligosaccharidic fractions are of the type obtainable by a processwhich comprises the steps of

contacting heparin (or heparinic fractions) possessing anticoagulantactivity and having chains with molecular weights ranging from about2000 to about 50000, with an agent capable of depolymerizing orfragmenting the heparinic chains, the conditions used for carrying outthat step being adjusted so as to obtain a depolymerization mixturewhich contains oligosaccharidic fragments or chains, constituted by nomore than 8 moieties yet having an anti-Xa Yin-Wessler activity andincluding a sequence consisting of less than 8 moieties, which sequenceis responsible, to a large extent, for the specific anti-Xa activity ofthe products;

treating the depolymerization mixture to separate at least the majorpart of the above defined oligosaccharidic chains, said treatmentadvantageously comprising (a) the contact of the depolymerizationmixture with ATIII for selecting at least the major part, advantageouslypractically the totality of the oligosaccharides possessing a sequencehaving the specific structure necessary to recognize and bind ATIII, (b)the elimination of the unselected products, (c) the recovery of theselected products. Said oligosaccharides can be characterized by thefact they possess a specific structure capable of binding ATIII. Theyhave an anti-Xa activity higher than heparin and a very low globalanticoagulant activity.

The products having an ATIII affinity as recovered from the abovementioned process, are submitted to one or several steps in order toselectively separate the above-defined oligosaccharides having shortchains.

.Iadd.BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elution diagram of a depolymerization heparin mixtureaccording to Example 2 of the present invention fractionated by gelfiltration on a DEAE Sephadex A 25 column.

FIG. 2 is an elution diagram of fractions obtained by filtration of theunfixed and fixed products of Example 3 of the present invention oncolumns of Sephadex G-50 Superfine gel.

FIG. 3 is an elution diagram of degraded FPA oligosaccharide chainsfragmented into di and tetrasaccharides according to the presentinvention wherein the di- and tetrasaccharides are separated bychromatography on a column of Sephadex G-50.

FIG. 4 is a graphical representation of anti-factor Xa activehexasaccharide fraction obtained by chromatography of depolymerizedheparin products on a column of Sephadex G-50 Superfine gel.

FIG. 5 is the ¹³ C NMR characterization of an octasaccharide fractionaccording to Example 3 of the present invention.

FIG. 6 is the ¹³ C NMR characterization of an octasaccharide fractionaccording to Example 1 of the present invention..Iaddend.

DESCRIPTION OF THE INVENTION

Said separation is advantageously obtained by fractionating the mixtureof the eluted products having ATIII affinity, according to theirmolecular weight and/or their ionic density, and recovering the desiredfractions.

Alternatively, the fractionation can be carried out directly on thedepolymerization mixture. At that stage, the fractions comprising shortoligosaccharidic chains, advantageously the oligosaccharides per se, canbe isolated. The fractions, optionally the oligosaccharides, with a highATIII affinity are subsequently separated by resorting to a stepcomprising contacting said fractions as above mentioned with ATIII.

The oligosaccharidic fractions of the invention are of the type of thoseobtained by resorting to the various steps defined above. They arecharacterized by the fact that they are free of oligosaccharidescomprising more than 8 saccharidic moieties and that they consist ofoligosaccharides having no more than 8 saccharidic moieties containing asequence comprising less than 8 saccharidic moieties, responsible fortheir anti-Xa activity at least to a major extent.

According to an embodiment, the fractions of the invention are of thetype obtainable by a process wherein the heparinic material isdepolymerized by a chemical process, in particular with nitrous acid,HNO₂, in aqueous medium.

The heparinic chains are thus split between a N-sulfate glucosamine unitand the following uronic acid resulting in chains containing a2,5-anhydromannose group at their reducing end.

In another embodiment, the depolymerization is carried out under anenzymatic process, advantageously with a highly purified heparinase,more especially a bacterial heparinase.

The enzyme cleaves the heparinic chains between the anomeric carbon ofan N-sulfate-glucosamine residue and the following uronic acid unit.

Its action produces a mixture of oligosaccharides(di-,tetra-,hexa-,octasaccharides) with a two fold degree ofpolymerization and terminated at their non reducing end by anα,β-unsaturated uronic acid.

The depolymerization with the purified heparinase is advantageouslycarried out under conditions giving rise to said biologically activeoligosaccharides having no more than 8 saccharide moieties and for partof them of 6 or less. Said conditions are such that the limit of theenzymatic reaction is reached. In other words the resultingoligosaccharides are then no longer susceptible to the action of thehighly purified heparinase.

The biologically active oligosaccharides then correspond to thoseseparable from said depolymerization mixtures (obtained by chemical orenzymatic way) by adsorption on carrier bound ATIII such as agarosebound ATIII under conditions enabling the products having an ATIIIaffinity to be fixed or retained on ATIII while those devoid of such anaffinity are eliminated for example by rinsing.

This is followed by the elution of the retained or adsorbed products inorder to recover them, and by their fractionation for isolating theshort chains having an anti-Xa activity.

Optionally, before said separation based on the AT III affinity of thefractions, the depolymerization mixture is submitted to a fractionationas already mentioned, to separate the desired chains. Such a separationis advantageously carried out by gel permeation (gel filtration).

Most of the oligosaccharidic chains such as those above isolated,besides their high AT III affinity and subsequent anti-Xa activity arecharacterized by NMR spectra comprising among others a characteristicsignal in the region of the C-2 of N-sulfate-glucosamine residues (saidsignal is marked by an asterisk on FIGS. 5 and 6). This signal does notappear with heparin. Very likely it can be related to the presence of a3-O substituent and more particularly to a 3-O-sulfate group on aN-sulfate-D-glucosamine moiety.

The oligosaccharides are further characterized by a Yin-Wessler titerand a USP titer which are respectively in a ratio of at least 30,preferably of at least 100.

Preferred oligosaccharidic fractions and oligosaccharides comprise thosewith an AT III affinity and an anti-Xa (Yin-Wessler) activity which arehigher than heparin. Such an anti-Xa activity can be 10 times as high asthe one of heparin. Activity higher than 100 iu/mg can be noticed forcertain oligosaccharides. Values above 700 ui/mg or of at least 1,000iu/mg, even 1,200, reaching 2,000 iu/mg or more have been observed forother oligosaccharides.

Active oligosaccharides of the invention have a N-sulfated D-glucosamineresidue advantageously 3-O sulfated (designated F in the formula givenhereafter).

Other oligosaccharides further comprise a N-acetyl-D-glucosamine residue(D) and/or a D-glucuronic acid residue (E) and/or a 2-Osulfate-L-iduronic acid residue (G) and/or a N-sulfate-D-glucosamineresidue (H).

In other oligosaccharides, the presence of the following saccharidicmoieties is observed i.e. a 2-O sulfate,4,5-unsaturated uronic acid (A)and/or a N-sulfate D-glucosamine unit (B) and/or a L-iduronic acid unit(C).

Preferred oligosaccharides of the invention have aN-sulfate-D-glucosamine residue at their reducing end. This residue issulfated or not in positions 3 and/or 6.

Some of the oligosaccharides contain all the residues hereaboveindicated.

The dosage by colorimetry of N-acetyl-glucosamine units, according tothe method of Smith and Gilkerson in Annal. Biochem. 1979,98, p.478-480, shows then the presence of about one molecule ofN-acetylglucosamine by oligosaccharidic chain. Glucosamine dosage beforeand after acid hydrolysis allows for determination of the repectiveamount of N-sulfate and N-acetyl-glucosamine.

Some oligosaccharides of the invention appear thus to be characterizedby the presence of one N-acetyl-D-glucosamine moiety for twoN-sulfate-glucosamine moieties.

Other oligosaccharides are characterized by the presence of oneN-acetyl-D-glucosamine moiety for three N-sulfate-D-glucosaminemoieties.

An oligosaccharide of that type is constituted by an octasaccharidecorresponding to the following ABCDEFGH sequence: ##STR1##

In the above formula R represents an hydrogen atom or a sulfate (SO₃ ⁻)group.

Another octasaccharide has ABGHCDEF sequence.

Active hexasaccharides of the invention also include moieties among saidA,B,C,D,E,F,G and H moieties. One of these species has ABCDEF sequence.

Another species has CDEFGH sequence wherein C is an unsaturated uronicacid.

Another species is constituted by CDEFGH sequence (but where C is aniduronic acid residue).

Other active oligosaccharides are pentasaccharides, more particularlythe one having DEFGH structure.

Shorter oligosaccharidic chains still biologically active are part ofthe present invention.

The pharmaceutically acceptable salts of said oligosaccharides are partof the invention as well.

The invention also aims at providing a process for obtaining fractionssuch as above disclosed, comprising heparin depolymerization and thetreatment of the resulting depolymerization mixture so as to separatethe fractions containing oligosaccharides having no more than 8saccharidic moieties (advantageously the oligosaccharides per se) havinga high AT III affinity and a high anti-Xa (Yin-Wessler) activity.

The depolymerization step is carried out under mild conditions so thatthe oligosaccharidic chains are not completely degradated and themoieties which are responsible (to a large extent) for anti-Xa(Yin-Wessler) activity are maintained.

Preferably, the depolymerizing conditions of the process definedhereabove are so adjusted as to maintain the capability of at least apart of the resulting oligosaccharides of being specifically retained onimmobilized antithrombin III.

The above adjustment can also advantageously be based on the upkeepingof the capability of at least part of the resulting oligosaccharides toexhibit anti-Xa activity as measurable by the Yin-Wessler test, whilehaving practically no more anticoagulant activity as measured by the USPtest.

It will be appreciated that the threshold with respect to the adjustmentof the depolymerization conditions need not necessarily be attained. Itis nevertheless desirable to do so when high yields of oligosaccharideshaving Yin-Wessler anti-Xa activity are desired.

When depolymerizing heparin with HNO₂, it seems advantageous to carryout the reaction in aqueous medium, at a pH ranging from 2 to 4,preferably 3 and at a temperature close to ambient.

When depolymerization with heparinase, a highly purified heparinaseshould preferably be used, in particular a bacterial heparinase, moreespecially originating from Flavobacterium heparinum. The conditions arecontrolled in order to obtain the smallest fragments still having anaffinity for AT III and an anti-Xa (Yin-Wessler) activity.

It is advantageously carried out at a pH ranging from 6 to 8, inparticular close to neutral and at a temperature close to ambient. Theheparinase is gradually added in the reaction mixture until hydrolysisis over.

The depolymerization with the heparinase may optionally be carried outon the fractions resulting from the oligosaccharidic fractions obtainedafter degradation of heparin with HNO₂, or also on the oligosaccharidesof higher molecular weight which are retained on immobilized AT IIItogetherwith the active oligosaccharides having less than 8 moieties andwhich are then eluted with these oligosaccharides having shorter chains.

The separation and the recovery of the fractions which contain thedesired oligosaccharides is advantageously carried out by affinitychromatography in a column containing bound AT III.

Satisfactory results are obtained by using a gel of agarose such as theone commercialized under the trademark Sepharose, with ATIII moleculeslinked thereon.

To achieve the desired separation of the major part of the productscontained in the depolymerization mixture and having a high anti-Xaactivity, the column is advantageously equilibrated with a buffer havingan ionic strength of about 0.2 M, preferably not less than 400.1 M, at apH of 6 to 8, preferably close to or slightly higher than neutral.

The products devoid of or having a low affinity for AT III areeliminated by rinsing with a buffer advantageously of the same type asthe one used for equilibrating the column.

A preferred embodiment for recovering the AT III retained or absorbedproducts, having an anti-Xa activity (Yin-Wessler) comprises desorbingand recovering all of the oligosaccharides by eluting them with a bufferhaving a sufficient ionic strength to that effect. The buffer used forthe above mentioned elution is further advantageously selected amongthose which do not interfere with the subsequent recovery steps(particularly with alcohol precipitation) of the oligosaccharidescontained in the recovered fractions. A buffer containing a calciumsalt, such as calcium chloride that remains soluble in presence of analcohol concentration (any suitable alcohol, for instance ethanol) whichcauses the precipitation of the oligosaccharides, is thus preferred.

A sodium salt can also be used.

After elution of the products having an AT III affinity, an alcoholicprecipitation is advantageously carried out to recover them, theirseparation being then made for example by centrifugation.

In order to have oligosaccharidic fractions having the required highanti-Xa activity (Yin-Wessler) and a satisfactory homogeneity, themixture of all the oligosaccharides previously retained on AT III isfractionated by a gel filtration or by ion exchange chromatography orboth or by any other method which would yield similar results.

Advantageously, the last mentioned fractionation is so monitored thatafter the larger molecules have been eluted, the smaller ones arerecovered, starting from those fractions which have a Yin-Wessler titerand a USP titer which are in a ratio of at least 30, preferably 100, therecovery extending to all the remaining fractions which still exhibitanti-Xa activity at least in the Yin-Wessler test.

Depending upon the amounts of solution subjected to gel filtration orchromatography, the volume of the successively eluted fractions will beselected as a matter of routine in order to select the most appropriatefractions with respect to the desired application.

Alternatively before the step of fixation on AT III, theoligosaccharides having more than 8 saccharidic moieties are eliminatedfrom the depolymerization mixture, advantageously by gel filtration or asimilar process as indicated above.

Biologically active octa-, hepta-, hexa-, penta-, tetra- andtrisaccharides can be isolated from the eluted fractions obtained.

The pharmacological study of the fractions and oligosaccharides of theinvention has shown a relationship between their anti-Xa activity asexpressed in Yin-Wessler units and their antithrombotic activity.

Said fractions and oligosaccharides appear then capable of exerting astrong antithrombotic activity. Due to their low or nil globalanticoagulant activity, the risks of hemorrhages are advantageouslypractically eliminated.

Among the assays made in vivo in order to study their antithromboticactivity, the following test has been carried out on the rabbit.

The formation of thrombus has been caused in the jugular vein of therabbit by injecting a complex of activated prothrombin.

The ability of ABCDEFGH octasaccharide having a Yin-Wessler titer of2000 units/mg to avoid the formation of the thrombus has been studied byinjecting the octasaccharide before the injection of 25 units/kg ofthrombogenic complex.

When injecting the oligosaccharides before the thromboplastin complex atdoses of 150 to 250 iu/mg Yin-Wessler, a significant fraction isobtained with respect. to the formation of the thrombus.

Said octasaccharide appears then to have a strong antithromboticactivity. Advantageously, global anticoagulant activity is notdetectable.

The oligosaccharide fractions according to the invention are free oftoxicity. The administration of 10,000 U/kg (Yin-Wessler titer) of anyof the fractions according to the invention causes in the rabbit neitherany toxic reaction nor any pyrogenic effect in the pyrogenicity test inthe rabbit according to the French Pharmacopea.

The compositions according to the invention are then particularlysuitable for controlling specifically some steps of the coagulation inman or animal, more particularly when selective control of the activityof the blood factor Xa is desired (by way of example only, in patients,who are to undergo or who underwent surgery, in atheromatous diseases,perturbations of the coagulation mechanisms by bacterial or enzymaticactivators, etc . . . )

The invention relates then to pharmaceutical preparations which containoligosaccharidic fractions or the oligosaccharides themselves with highanti-Xa activity.

It relates more particularly to pharmaceutical preparation devoid ofpyrogenic substances, containing an effective amount of activeprinciples, in association with pharmaceutical excipients.

Particularly it concerns the compositions in which the pharmaceuticalvehicle is suitable for the administration by oral route. Dosage formsof the invention suitable for the administration by oral route canadvantageously be gastroresistant capsules, or tablets or pills.

Other pharmaceutical compositions include oligosaccharides oroligosaccharidic fractions in association with excipients suitable forthe administration by rectal route. Corresponding dosage forms aresuppositories.

Other dosage forms of the invention are sprays or ointments.

The invention also concerns pharmaceutical compositions which areinjectable, sterile or sterilizable.

Such solutions advantageously contain 1,000 to 100,000 U (Yin-Wessler)mlof the oligosaccharidic fraction, preferably from 5,000 to 50,000 forexample 25,000 U/ml, when these solutions are intended for subcutaneousinjection or containing again, for example, from 500 to 10,000 forexample, 5,000 units/ml of the oligosaccharidic fraction oroligosaccharide when they are intended for intravenous injection or forperfusion.

Advantageously, these pharmaceutical preparations are presented in theform of non-reusable syringes, ready for use at any suitable time.

The invention also concerns pharmaceutical compositions containing saidoligosaccharides in association with another active principle, inparticular useful for the prophylaxis and the treatment of thrombosissuch as a veinotonic agent like dihydroergotamine, a salt of nicotinicacid or a thrombolytic agent like urokinase.

The oligosaccharidic fractions and oligosaccharides of the invention areadvantageously in the form of a salt of at least one physiologicallyacceptable metal, such as sodium and/or calcium and/or magnesium.

The pharmaceutical compositions according to the invention areparticularly adapted to the control (preventive or curative) of theblood coagulation in man or animal, notably in those cases where thehost is subjected to risks of hypercoagulability, more particularlythose resulting from the release by the organism of thromboplastin, forexample, of tissular thromboplastin (surgical operations, atheromatousprocesses, tumor development, disturbances of the coagulation mechanismsby bacterial or enzymatic activators, etc . . . ). For the sole purposeof illustrating the invention, and without being a cause for limitingthe protection of the invention, there will be indicated below, by wayof example, a posology capable of being used in man: it comprises forexample, the administration to the patient of 1,000 to 25,000 U by thesub-cutaneous route, 2 to 3 times daily, according to the level ofhypercoagulation risk or the thrombotic condition of the patient, orfrom 1,000 to 25,000 U per 24 hours by the intravenous route, indiscontinuous administration at regular intervals or continuously byperfusion, or again from 1,000 to 25,000 U (three times weekly) by theintramuscular route (titers expressed in Yin-Wessler U). The dosesshould naturally, be adjusted in each patient according to the resultsof previously effected blood analyses, the nature of the disorder fromwhich the patient is suffering and, generally, his state of health, asis well known.

The invention also relates to the application of the oligosaccharidesaccording to the invention for the preparation of a biological reactantusable in the laboratory, notably as a comparison reference for thestudy of other substances of which the anticoagulant activity is to betested, notably at the level of inhibition of the factor Xa.

The description hereafter of examples of production of theoligosaccharides having YIN and WESSLER anticoagulant activity accordingto the invention will be given for the sake of further illustrating, yetin a nonlimitative manner, the invention.

EXAMPLE 1

Method for obtaining oligosaccharidic fractions including the steps of:

I. depolymerizing heparin with HNO₂,

II. separating the biologically active oligosaccharides bychromatography on Sepharose-AT III,

III. gel filtration of the eluted fractions and recovery of the desiredproducts.

I. Depolymerization of heparin in the presence of nitrous acid

20 g of heparin (USP titer: 150 UI/mg, YIN-WESSLER titer: 150 U/mg) weredissolved in 800 ml of water at room temperature. 200 ml of 0.5 Msulfuric acid and then 13.8 g of sodium nitrite were then added (finalmolarities of sulfuric acid: 0.1 M, and of the nitrite: 0.2 M). Areaction accompanied by the release of gaseous nitrogen was thenproduced. The reaction was stopped 15 minutes later by adjusting the pHat 7-7.2 with 5 N sodium hydroxide. The depolymerization products wereprecipitated by ethanol (7 volumes) that is, in this case, 7 liters. Theprecipitate was centrifuged, washed with alcohol and dried under vacuum.

    ______________________________________                                        Weight                 24.8 g                                                 USP titer              nil                                                    YIN-WESSLER titer      7 μ/mg                                              ______________________________________                                    

The apparent increase of weight of the precipitate was due to partialprecipitation of sodium sulfate together with the depolymerizationproducts.

II. Chromatography on SEPHAROSE-antithrombin III

The latter depolymerization products were then submitted to achromatography on a SEPHAROSE gel comprising antithrombin III retainedthereon. A column (diameter 2.6 cm, height 40 cm) containing 200 ml ofSEPHAROSE-AT III (approximately 10 mg of immobilized bovin antithrombinIII per ml of SEPHAROSE) was equilibrated with a buffer consisting ofNaCl 0.2 M. tris-HCl 0.05 M pH 7.2.

1.6 g of the abovesaid depolymerization products were dissolved in 16 mlof 0.2 M NaCl buffer and percolated through a column at a flow-rate of50 ml/h and the column was rinsed by 500 ml of 0.2 M NaCl buffer.

The retained oligosaccharides were then eluted from the column by abuffer capable of desorbing all of the oligosaccharides retained on theimmobilized antithrombin III (CaCl₂ 0.2 M, tris-HCl 0.05 M, pH 7.2). Thepart of effluent which contained the oligosaccharides was recovered andthe said oligosaccharides were precipitated by 10 volumes of ethanol.

After washing with ethanol and drying under vacuum, 5 mg of products arerecovered having a USP titer of 66 UI/mg and a YIN and WESSLER titer of1,600 U/mg.

The products so obtained consisted of a mixture of mucopolysaccharidesof high molecular weight and of oligosaccharides of low molecularweight.

III. Gel filtration

90 mg of a mixture of mucopolysaccharides and oligosaccharides obtainedby a procedure of which the two preceding paragraphs are representativewere dissolved in 2 ml distilled water, then deposited on the top of acolumn of the filtration gel formed of extremely fine particlescommercialized under the denomination SEPHADEX G 50 (height 1 m,diameter 2.6 cm), equilibrated with distilled water. Development of thecolumn with distilled water was carried out under a flow-rate of 24ml/h. The effluent was recovered fraction-wise (6 ml per tube). Themucopolysaccharide content of each tube was evaluated by UV absorptionat 206 nanometers.

The contents of the tubes were pooled to form the fractions numbered 1to 5.

The mucopolysaccharides contained in each of these fractions wereprecipitated by alcohol and dried.

The weights and anticoagulant properties of these fractions were asfollows:

    ______________________________________                                        fraction (1) weight          15     mg                                                     USP titer       156    μl/mg                                               YIN-WESSLER titer                                                                             190    μ/mg                                   fraction (2) weight          16     mg                                                     USP titer       139    μl/mg                                               YIN-WESSLER titer                                                                             520    μ/mg                                   fraction (3) weight          17     mg                                                     USP titer       50.3   μl/mg                                               YIN-WESSLER titer                                                                             1 390  μ/mg                                   fraction (4) weight          40     mg                                                     USP titer       7      μl/mg                                               YIN-WESSLER titer                                                                             870    μ/mg                                   fraction (5) weight          1      mg                                                     USP titer              nil                                                    YIN-WESSLER titer                                                                             150    μ/mg                                   ______________________________________                                    

The ranges of molecular weights of the fractions so obtained, moreparticularly of the abovesaid "fraction 4" and "fraction 5" wereappreciated both by paper chromatography and High Pressure LiquidChromatography (HPLC) and on a comparative basis with the resultsobtained in the same experimental systems with a decasaccharide ofheparin and:

in paper chromatography, with the reference products disclosed in thearticle of SILVA and DIETRICH (SILVA M. E. et DIETRICH C. P., J. Biol.chem. Vol. 250 (1975) pp. 6,841-6,846, and

in HPLC on a silica gel, with a series of polystyrene-sodium sulfonatesof increasing known molecular weights and heparin samples of knownmolecular weights.

"Fraction 4" was found to consist of an oligosaccharide having less than8 saccharide units and most likely not more than 6 saccharide units, and"fraction 5" of an oligosaccharide having less than 6 saccharides, mostlikely not more than 4 saccharide units.

EXAMPLE 2

Method for obtaining oligosaccharidic fractions including the steps of:

A.--depolymerizing heparin by digestion with an heparinase;

B.--fractionnating the depolymerization mixture by gel filtration onDEAE Sephadex A 25;

C.--selecting the biologically active products by chromatography onagarose AT III.

A. Depolymerizing heparin by digestion with an heparinase

1. Preparation of the heparinase

Flavobacterium heparinum enzymes were used for degradating heparin.

The enzymes of flavobacterium heparinum were cultivated according to themethod of Payza and Korn in J. Biol. Chem. 1956, 223, p. 853-858. Thelyophilized cells were crushed under dry conditions in the presence ofalumine and extracted by an acetate buffer at neutral pH.

The insoluble parts were eliminated and the solution was successivelychromatographied on diethylaminoethyl cellulose and then on two agarosessuch as those commercialized under the trademarks CM Sepharose CL 6B andUltrogel ACA 54 respectively.

20 mg of heparinase having a degree of purity of 90% (the purity havingbeen evaluated by electrophoresis) and a titer of 30,000 units/mg (8,333units as measured by the method of HOVINGH et al (1970), J. Biol. Chem.245 6, 1970) were thus obtained.

2. Depolymerization of heparin

1. g of heparin for use in therapy was dissolved in 50 ml of distilledwater. 1 g of sodium acetate and 100 mg of calcium chloride were addedthereto. The pH was adjusted to 7.2 by ClHO. 1 N.

1. mg of a solution of highly purified bacterial heparinase having atitre of 30,000 units/mg (8,333 units as measured by the method ofHOVING et al (1970), J. Biol. Chem. 245, 6, 170) was added and themixture was incubated for 15 hours at 30° C. After precipitation by 10volumes of ethanol at 100° GL the depolymerized product obtained wasdried. The product obtained was designated as P.

B. Fractionation on "DEAE SEPHADEX A 25"

A column having a 16 mm diameter containing 50 ml of the gel known underthe above commercial designation was equilibrated with a 0.1 M NaCl, pH7.0 buffer. 20 ml of a solution of 180 mg of the above product P in saidbuffer were deposited on the top of the column and eluted with agradient formed starting from 250 ml of a 0.1 M NaCl, pH 7.0 buffer, onthe one hand, and of a 0.5 M CaCl₂ pH 7.0 buffer, on the other hand. Theflow rate was adjusted to 30 ml per hour. Successive 10 ml volumes wererecovered.

The elution diagram (followed by optical density measurements at 232 nm)is represented on FIG. 1. The volumes were pooled into fractions 1, 2,3, 4 according to the optical density measurements, as shown in FIG. 1.Their respective optical densities are shown in table 1 hereafter.

                  TABLE I                                                         ______________________________________                                                                    α!.sub.D .sup.b (c = 1,                                   Yield in weight                                                                            water                                              No            %            On the  DO..sub.H2O .sup.232 nm                    frac-         from 120.8 mg of                                                                           D band  (conc:                                     tion  Weight  starting product                                                                           of sodium                                                                             1 mg/ml                                    ______________________________________                                        1     22 mg   18%          +33°                                                                           2.1                                        2     69 mg   57%          +30°                                                                           3.2                                        3     20 mg   16%          +30°                                                                           2.6                                        4     9.8 mg   8%          +21°                                                                           1.7                                        ______________________________________                                    

C. Affinity chromatography on agarose AT III of the above secondfraction

A column having a diameter of 2.5 cm containing 50 ml of agarose havingAT III immobilized thereon (Agarose AT III) was equilibrated with 0.1 M.NaCl tris-HCl 0.025 M; pH 7.4.

60 mg of the fraction numbered 2 in table I (P 2) dissolved in 6 ml ofthe above buffer were deposited on the top of the column. Elution wascarded out with successive solutions.

The first eluant used was the same as above: an effluent containing 50mg of a product referred to as P 2 (B) contained in the first detectedpeak of optical density at 232 nm was separated therewith.

On changing the eluant to 0.2 M NaCl, 0.05 M tris HCl; pH 7.4, no activefraction was desorbed from the gel.

On further changing the eluant to 2 M CaCl₂ a peak was obtained. Thevolumes containing the fractions which gave rise to said peak wereprecipitated by alcohol to provide 5 mg of a highly active fractionaccording to the invention (referred to as P 2(A).

The biological and physical characteristics of the last mentionedfraction are indicated herebelow:

    ______________________________________                                        YIN-WESSLER titer; 1 400 units/mg                                              α!.sub.D .sup.20 = +24° (c = 1, water)                          DO..sub.HCl 0.03 N .sup.235 nm = 4.5                                          ______________________________________                                    

The analytical results concerning P 2 (A) fraction are as follows:

1. Chemical analysis

The components of P 2 (A) were dosed as concerned:

the uronic acids, according to the method of Bitter T. et al. Anal.Biochem., 4, (1962), 330-334;

the hexosamines, on the one hand by the Elson-Morgan method, as modifiedby Antonopoulos C. A. et al., Biochem. Biophys. Acta 83, (1964), 1-19(after hydrolysis of P 2 (A) by 6 N HCl for 4 hours, at 110° C. in anitrogen atmosphere) and, on the other hand, by the method of Smith R.L. and Gilkerson E., Anal. Biochem., 98, (1979), 478-480, on samples ofeither non-hydrolysed, or hydrolysed samples of P 2 (A);

the sulfates, by the method of T. T. Terho et al., Anal. Biochem., 41,(1971), 471-476.

The results as to composition are summed up in table II hereafter,expressed as weight % (first line), as the mole to mole ratio withrespect to the glucosamine units after hydrolysis (H⁺) according to thetechnique of Smith and Gilkerson (second line).

                  TABLE II                                                        ______________________________________                                                   Hexosamines                                                                     Smith-Gil.                                                       Uronic acids H.sup.(+)  Antonopoulos                                                                             Sulfates                                   ______________________________________                                        P 2 (A)                                                                              15,2%     18,7%      12,8%    12%                                             0,9       1,0        0,7       1,4                                     ______________________________________                                    

The results show that the oligosaccharides of the invention comprisesubstantially:

1. mole of uronic acid per 1 mole of hexosamine;

1.4 mole of sulfate per disaccharidic unit (including an uronic unit andan hexosamine unit);

2 N-sulfate-glusosamine units, per 1 N-acetylglusocamine units.

2 High Pressure Liquid Chromatography

50 μl of a solution of 0.5 mg/ml of P 2 (A) were subjected to an elutionby 0.02 M Na₂ SO₄ (1 ml/minute) through a series of a first and secondcolumns (250×4.6 cm) respectively filled with silica commercializedunder the designation LICHROPHOSPHER Si 100, and a third column (250×3cm) of the material commercialized (by WATERS and Associates) under thedesignation micro-BONDAGEL. The molecular weight of the fractionaccording to the invention was approximated by its time of elution (ofof retention on the columns) with reference to the linear variations ofthe retention times of standard polystyrene-sulfonates of knownmolecular weights (of respectively 4,000, 6,500, 16,000 and 31,000) aswell as of a tetrasaccharide having a known molecular weight of 700-900subjected to the same assays, as a function of the logarithm valuescorresponding to said molecular weights. The measurements were madeusing a SPECTRAPHYSICS 3500 chromatograph, and the detection of thefraction by UV spectrophotometry (200 mμ and 230 mμ).

This assay, as well as an other chemical assay according to the methodA. Linker and P. Hovingh, Biochem. vol. 11, No. 4, 1972, p. 563-567,confirmed the fact that the active oligosaccharide contains not more,and even less than 8 saccharidic units.

3. Ultra-violet absorption

UV-absorption was carried out on a solution of 100 γ/ml of P 2 (A) andin an UV band of 215-260 nm. Maximum absorption was observed in the230-235 nm band.

EXAMPLE 3

Method for obtaining oligosaccharidic fractions including the steps of:

I. enzymatic depolymerization of heparin;

II. selecting the biologically active products by chromatography onSepharose AT III;

III. gel filtration of the eluted fractions and recovery of the desiredproducts.

1. Enzymatic depolymerization of heparin

20 mg of heparin for use in therapy were dissolved in 100 ml of anacetate buffer (NaOAc 0.15 M, NaCl 0.15 M; Ca Cl₂ 0.005 M; pH 6.9).

The solution was incubated for 24 hours at 30° C.

The highly purified heparinase previously obtained was added to thesolution as follows:

0.4 mg (amount based on the dosage of the proteins) at the time t=0,

0.2 mg at the time t=+8 hours,

0.2 mg at the time t=+24 hours

At t=+36 hours, another addition of heparinase did not cause an increaseof the optical density at 232 nm. The reaction was considered as endedand the products were recovered by alcoholic precipitation and weredried (yield in weight 90%).

2. Affinity chromatography on Sepharose AT III of the depolymerizedproducts

A chromatography column (5×18 cm) containing 10 mg of bound AT III/ml ofSepharose was equilibrated with 0.1. M Na Cl; tris-HCl 0.05 M; pH 7.5. 2g of the products of degradation of heparin were deposited on the top ofthe column. The column was rinsed with said buffer whereby the unfixedfractions were eliminated (the corresponding products being designatedas UP hereinafter). The fixed products (designated as F P-were theneluted with a solution of CaCl₂ 1 M pH 7.2. The products wereprecipitated with alcohol and recovered by centrifugation. 1.6 g UP onthe one hand and 8 mg of FP having 800 ui/mg (Yin-Wessler), on the otherhand are thus recovered.

3. Gel filtration

UP and FP products were subjected separately to gel filtration 25 mg ofeach product were filtrated on a column. (200×0.6 cm) of a Sephadex G 50Superfine gel. The elution was carried out using a solution of NaCl 0.2M Fractions of 0.65 ml were collected. After elimination of the salts onSephadex G 25, the products were lyophilized, FIG. . .3.!. .Iadd.2.Iaddend.represents the elution diagrams of UP fractions (continuousline) and FP (fractions (dotted line). The elution has been controlledby optical density measurements at 230 nm (absorption wavelength of thedouble bond created by the heparinase).

According to the optical density measurements, UP fraction wasfractionated into di-, tetra-, hexa- and octasaccharides (UP₂, UP₄, UP₆and UP₈) and into another product having a higher polymerization degree(UP₊₈). UP₈ and UP₊₈ do not appear on the elution curve of FIG. 2 asthey only represent a very low percentage (3.4 and 1.3% respectively)and are only detected at high sensitivity. FP fraction was separatedinto four fractions designated FPa,b,c and d, according to the order ofthe decreasing elution volume. The major part of FP fractions is elutedslightly before UP fraction.

Results concerning each of UP and FP fractions are given in the tablehereinafter.

They comprise: the elution volume (ml) of each of said fractions, issuedfrom UP and FP fractions; the % of each of them in the mixture of the UPand FP fractions respectively, the specific rotatory power of eachfraction (measured with an electronic polarimeter in aqueous solutionsusually 1%); the absorbance at 230 mn (measured in a 0.01% solution inHCl 0.03 M, the results being expressed as optical density of 1%solution); and the anti-Xa activity in human plasma of FP fractionsmeasured according to the test of Yin and Wessler above mentioned.

                                      TABLE                                       __________________________________________________________________________        Elution                                                                       Volume                                                                            % in the                                                                           α!.sub.D .sup.20 (c = 1, water)                                                          Activity anti-Xa                                Fraction                                                                          (ml)                                                                              mixture                                                                           on the D band of sodium                                                                  DO..sub.H2O .sup.230 nm                                                              μl/mg Yin-Wessler                            __________________________________________________________________________    UP 2                                                                              52-60                                                                               52                                                                              +5         6      --                                              UP 4                                                                              47-52                                                                             30,5                                                                              +23,5      4,8    --                                              UP 6                                                                              43-47                                                                               13                                                                              +37        3,3    --                                              UP 8                                                                              40-43                                                                               3,4                                                                             +41,5      2,5    --                                              UP + 8                                                                            37-40                                                                               1,3          2                                                      FPa 40-45                                                                               50                                                                              +39        3,2    1200(2000 in another te)                        FPb 35-39                                                                               23                                                                              +41        2,5    930                                             FPc 27-34                                                                               14                                                                              --         --     200                                             FPd 23-26                                                                               5 --         --     330                                             __________________________________________________________________________

It appears from the results given in the table that each of the FPafractions exibit an anticoagulant activity, FPA having the mostimportant biological activity. It will be noted that it represents 50 to75% of the FP mixture. The FPa fraction was submitted to varioustreatments in order to elucidate its structure. The following analyticalresults were obtained.

Degradation of FPa by treatment with nitrous acid

The FPa fractions recovered after the gel filtration were incubated withHNO₂ in an aqueous medium under conditions enabling degradation of theoligosaccharidic chains. The degradation was carried out according tothe method of Shiveley and Conrad in Biochemistry 1976, 15, p.3932-3942.

Under the action of NHO₂ the oligosaccharidic chains are fragmented intodi- and tetrasaccharides, the break taking place behind the N-sulfatedglucosamine moieties said moieties being converted into 2,5anhydromannose groups.

The di- and tetrasaccharides so obtained are separated by chromatographyof Sephadex G50 (200×0.5 cm; NaCl 0.2 M.) The elution diagram is givenon FIG. 3.

The following measurements are made on each fraction: the opticaldensity at 230 nm (curve a) the amount of uronic acids (curve b) of2,5-anhydro-mannose (curve c) and of glucosamine (before and after acidhydrolysis), the radio activity when the analysed products have beentritiated before being degradated by HNO₂.

The optical density measurement of these fractions shows that the majorpart of the unsaturated molecules are disaccharides, 90% or the opticaldensity being in the peak of the disaccharide while 10% are in the peakof the tetrasaccharidic fragment.

It can be considered therefrom that the tetrasaccharidic unit does notcontain an uronic acid moiety with a double bond. Such moiety is part ofa disaccharide which contains further an N-sulfoglucosamine, thisdisaccharide GH is located at the non reducing end of theoligosaccharide before its nitrous degradation.

Besides, by dosing 2,5 anhydro-mannose groups in these fractions (curvec in FIG. 3), 33% of 2,5 anhydromannose groups are found in thetetrasaccharidic fragments and 66% in the disaccharidic fragments. Byassuming that the oligosaccharidic chains are N-sulfoglucosamineterminated, it can be concluded from said results that FPa contains twodisaccharidic chains for one tetrasaccharidic chain (see curve b on FIG.3). Said results were confirmed by the dosage of the uronic acids (curveb) according to Bitter and Muir in Annal; Biochem. 1962,4 p. 330-334 andof the glucosamine moieties in the degradated products originating fromFPA, i.e. said degradated products comprise twice as many disaccharidicmolecules as tetrasaccharidic melocules.

Reduction of fraction FPa by sodium borohydride followed by nitrousdegradation

By reducing FPa before the nitrous degradation, the reducing ends of thechains are not converted into 2,5-anhydro-mannose during the nitrousdegradation. The reduction is carried out with a sodium borohydridebuffer at pH 9.5. The reducing ends of the FPa oligosaccharidic chainsare then converted into tritiated hexitols. The reduced product isseparated from the salts present in the mixture by filtration onSephadex G 25. It is then submitted to nitrous acid degradation and gelfiltration as described above. The dosage of 2,5-anhydro-mannose groupsis then carried out and shows a clear decrease of said groups in thedisaccharidic fraction while the tetrasaccharidic fractions remainpractically unchanged.

By reducing with tritiated borohydride, 70 to 80% of the radioactivityare found in the disaccharides and 20 to 30% in the tetrasaccharides.Furthermore, it was observed that after said treatment, the amount of2,5-anhydro-mannose decreases far more in the disaccharidic peak than inthe tetrasaccharidic peak--such result being in favour of the presenceof a disaccharidic fragment at the reducing end (70% of the molecules)and also of a tetrasaccharide (30%).

Nitrous acid degradation of FPa fraction under very mild conditions

The FPa fraction was further submitted to a nitrous acid degradationunder very mild conditions. It was thus possible to obtain, after gelfiltration and affinity chromatography an oligosaccharide fractioncontaining mainly the two hexasaccharides with ABCDEF and CDEFGHsequences the latest being also part of the invention.

The procedure involved treatment of the said FPa fraction with nitrousacid as described by Cifonelli and King (Carbohydrate Res. 1972, 21,pp.173-186), except that the reaction was stopped between 1 and 5 mn andpreferably 3 mn. The fragments thus obtained were desalted by gelfiltration on Sephadex G 25 and submitted to an affinity chromatographyin the same conditions as described above.

Two main species, i.e. CDEFGH and ABCDEF were found in the elutedfractions, as shown by the analytical methods described above for studyof the FPa fraction. They still had a high anti-Xa activity (Yin andWessler).

NMR characterization of an octasaccharidic fraction

The octasaccharidic fraction was isolated. It presented a YW titer of2000 IU/mg and an APTT titer of 4 IU/mg. The ¹³ C NMR spectrum of thisproduct was recorded (see FIG. 5). If confirms that the product is anoctasaccharide. It also confirms the assigned structure (ABCDEFGHsequence). The signals observed are respectively characteristic of the:

anomeric carbon in position 1 (90-105 ppm) of the various moieties(A,B,C,D,E,F,G and H) of the structure (the corresponding moiety beingmentioned for each peak to enable their identification); on the figure

carbons in position 6 (about 60 and about 70 ppm) (signal C₆) and inposition 2 (55-60 ppm) (signal C₂) of the glucosamine moieties;

CH₃ of --NHAc group in D (about 25 ppm). Furthermore the presence of anew resonance signal in the C-2 region of N-sulfo-amino-glucosamineresidue is observed (signal x--this signal does not correspond to anyresonance signal in NMR spectra obtained under similar conditions with aconventional heparin).

EXAMPLE 4 Isolation of an anti-factor Xa active hexasaccharide fraction

In another set of experiments, the mixture obtained after the affinitychromatography step (81 mg), was chromatographied on a column (200×2.5cm) of Sephadex G-50 superfine gel.

The elution was performed with 0.2 M sodium chloride. The products weredetected by U.V absorption at 232 nm (see FIG. 4).

The major part of the product was eluted in the octasaccharidic region,and presented the same properties as the product previously described.

The hexasaccharide fraction was collected and the salts were eliminated.The product thus obtained was freeze-dried. Then yield was 2 mg.

This compound was highly active in the YW test: 510 IU/mg. Its APTTtiter was 3IU/mg. Since this hexasaccharidic fraction is obtained afterheparinase degradation, it contains the residues A and H characterizedin the octasaccharidic fraction. Moreover, since this product presentsaffinity for ATIII it should contain a CDEF tetrasaccharidic sequence.Thus it can be represented by ABCDEF structure.

However one cannot exclude the presence, in this material, of smallamount of a product having CDEFGH structure, where C is an unsaturatedhexuronic acid residue (the 2-OH group being sulfated or not).

A study carried out as described for the octasaccharide (i.e. nitrousacid degradation and examination of the fragments) indicates thepresence of both ABCDEF and CDEFGH.

EXAMPLE 5 Degradation of ABCDEFGH octasaccharide and oligosaccharidesobtained

1--By using the method described by L. A. Fransson in CarbohydrateResearch 62,235-244, 1978 ABCDEFGH octasaccharide is submitted to anoxidation reaction with sodium periodate in phosphate medium, pH 7 at37° C. during about 14 hours. An alkaline hydrolysis is caused by addingNaOH to a pH of 11-12 and the reaction mixture is allowed to stay atroom temperature for 30 minutes. It is then neutralized; a trisaccharidewhich is likely to have a structure FGH is separated by chromatographyon Sephadex G 50. The determination of the anti-Xa activity according tothe Yin-Wessler test give a value about 100-200 ui/mg depending on theassay.

2--Said octasaccharide is submitted to the action of a purifiedheparitinase, extracted from Flavobacterium heparinum. The experimentalconditions (in particular, pH, temperature, time) correspond to thoseused in the enzymatic degradation according to example 2. Twotetrasaccharides to which structures EFGH and ABCD can be given arerecovered by filtration on SEPHADEX G 50 (carried out as in example 2).By passing the tetrasaccharides through a column of Sepharose ATIII,EFGH is be retained while ABCD is eliminated. EFGH is then recovered byelution with a solution NaCl 1 M. The Yin-Wessler activity, measured invarious assays, is about 100-200 ui/mg.

3--Octasaccharide ABCDEFGH is submitted to a mild nitrous degradation. 1mg of octasaccharide is used per ml of solution--HNO₂ is generated insitu by adding NaNO₂ N/1000 and Hcl. The pH is adjusted to 3. After 10mn, at the ambient, the pH is adjusted to 7. The reaction mixture issubmitted to a gel filtration followed by an affinity chromatography,using the conditions disclosed in the preceeding examples. By elatingwith NaCl 1 M or CaCl₂ 1 M, a product can be recovered having presumablyhexasaccharidic structure CDEFGH. Said product is submitted to theaction of an iduronidase extracted from human kidney. The enzymaticdegradation step is carried out at pH 7 at 37° C.

An oligosaccharide to which pentasaccharidic structure DEFGH isattributed is obtained by filtration of the depolymerization mixture onSephadex G 50. The anti-Xa (Yin-Wessler) activity of this productappears to be over 400 ui/mg.

EXAMPLE 6 NMR characterization of the oligosaccharidic fraction obtainedaccording to example 1

The ¹³ C NMR spectrum of this fraction shows the presence of saidsignal * in the region of C-2 of glucosamine residues (see FIG. 6). Thissignal can be assigned to a glucosamine unit which is N-sulfated andsubstituted by a --OSO₃ ⁻ group in position 3.

This glucosamine unit is sulfated or not in position 6.

Furthermore, the integral curve confirms that the product has an averagenumber of moieties of less than 8 and comprises a major part ofhexasaccharidic and octasaccharidic species. Starting from beef lungheparin, after controlled degradation, followed by affinitychromatography and gel filtration as described above an octasaccharidehaving the structure ABCFGHGH can be isolated. This product was highlyactive in an anti-Xa assay. An hexasaccharide ABCFGH was also extractedin minute amount. Ic was also active in an anti-Xa assay.

The invention extends of course to the oligosaccharides that may beobtained by other depolymerization techniques of heparin or hepariniccompounds followed by the subsequent recovery of the oligosaccharides oflow molecular weight having anticoagulant activity as measurable by theYIN-WESSLER test.

As an example of another depolymerization technique of heparin orrelated compounds, one may cite periodic oxidation. One may also citethe technique which consists in producing an α,β elimination reaction bychemical means on heparin or esters of heparin giving similar results,or the process which comprises:

contacting the starting heparin fraction with antithrombin IIIimmobilized on a Sepharose gel to fix the antithrombin-bindingcomponents of said heparin fraction,

digesting the so fixed heparin with a bacterial heparinase and

eluting the antithrombin-binding fragments from the gel whereby aneluant containing the above said fragments, all of which may then besubjected to the further steps of the above-defined process.

We claim:
 1. An oligosaccharide fraction of the heparin chain whicholigosaccharide has improved antithrombotic activity in vivo higher thanthat of heparin (as measured by the Yin-Wessler test), whicholigosaccharide fraction (1) comprises not more than 8 saccharide units,(2) of which one is an N-sulfate-3-O-sulfate-D-glucosamine unit (3) hasanti-Xa activity at least 10 times that of heparin, (4) specificaffinity for AT III, (5) a ratio of Yin-Wessler titer to USP titer of atleast 30 and (6) virtually no anticoagulant activity (as determined bythe USP test) and, the biologically acceptable salts of saidoligosaccharide.
 2. The oligosaccharide of claim 1 which oligosaccharidecontains a saccharide sequence of units selected from the groupconsisting of ABCDEFGH, ABGHCDEF, ABCDEF, CDEFGH and DEFGH, wherein A,B, C, D, E, F, G and H are defined as followsA=unsaturated or saturateduronic acid B=N-sulfate-D-Glucosamine orN-sulfate-6-O-sulfate-D-glucosamine C=L-iduronic acid or, where presentat a chain end, unsaturated uronic acid D=N-acetyl-D-glucosamine orN-acetyl-6-O-sulfate-D-glucosamine E=D-glucuronic acidF=N-sulfate-3-O-sulfate-D-glucosamine orN-sulfate-3-O-sulfate-6-O-sulfate-D-glucosamine G=2-O-sulfate-L-iduronicacid, and H=N-sulfate-D-glucosamine orN-sulfate-6-O-sulfate-D-glucosamine.
 3. The oligosaccharide of claim 1wherein the N-sulfate-D-glucosamine isN-sulfate-3-O-sulfate-6-O-sulfate-D-glucosamine.
 4. The oligosaccharideof claim 1 which includes a unit selected from the group consisting ofN-acetyl-D-glucosamine, D-glucuronic acid, 2-O-sulfate-L-iduronic acidor N-sulfate-D-glucosamine.
 5. The oligosaccharide of claim 1 whichincludes a unit selected from the group consisting of 2-O-sulfate-4,5unsaturated uronic acid, N-sulfate-D-glucosamine or L-iduronic acid. 6.The oligosaccharide of claim 1 which includes a unit selected from thegroup consisting of N-sulfate-6-O-sulfate-D-glucosamine, L-iduronicacid, 2-O-sulfate-L-iduronic acid or N-sulfate-D-glucosamine.
 7. Theoligosaccharide of claim 2 wherein the saccharide sequence is selectedfrom the group consisting of CDEFGH or ABCDEF.
 8. The oligosaccharide ofclaim 2 wherein the saccharide sequence is selected from the groupconsisting of DEFGH.
 9. The oligosaccharide of claim 1 which is thesodium, calcium or magnesium salt.
 10. The oligosaccharide of claim 1wherein the N-sulfate-D-glucosamine unit is at the reducing end of theoligosaccharide chain.
 11. The oligosaccharide of claim 1 whichcomprises one N-acetyl-glucosamine saccharide moiety for every two orthree N-sulfate-glucosamine saccharide moieties.
 12. A therapeuticantithrombotic composition which has antithrombotic activity higher thanthat of heparin (as measured by the Yin-Wessler test) which compositioncomprises a therapeutically acceptable carrier and in a therapeuticallyeffective amount, an oligosaccharide of claims 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or
 11. 13. The therapeutic composition of claim 12 in which theratio of Yin-Wessler titer to USP titer is at least about
 100. 14. Thetherapeutic composition of claim 12 in which the oligosaccharide has aYin-Wessler titer of 100 to about 2,000 U/mg.
 15. A therapeutic methodfor controlling thrombosis in a patient which comprises administering tosaid patient the therapeutic antithrombotic composition of claim 12 andcontrolling thrombosis.
 16. The therapeutic method of claim 15 in whichthe administration of the composition is at periodic intervals.